Nanoparticle probes and methods of making and use thereof

ABSTRACT

Some embodiments relate to nanoparticle probes for the detection of disease states in a patient or for tissue engineering. In some embodiments, the nanoparticle probe comprises one or more slip bonds that bind to a cell surface structure. In some embodiments, the binding of the nanoparticle probe is selective. In some embodiments, the nanoparticle probe binds to cells having a certain maximum glycocalyx thickness.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/680,016, filed Aug. 17, 2017, which is a continuation-in-part of U.S.patent application Ser. No. 15/461,035, filed Mar. 16, 2017, whichclaims the priority benefit of U.S. Patent Application No. 62/309,751,filed Mar. 17, 2016, all of which are hereby incorporated by referencein their entireties.

BACKGROUND Field

The present disclosure pertains to nanoparticle probes, methods ofmaking nanoparticle probes, and methods of using nanoparticle probes forbiological targeting.

Description of the Related Art

The glycocalyx layer covers the surfaces of various cell types and iscomprised of glycoproteins and other carbohydrate-based moieties.

SUMMARY

Some embodiments described herein pertain to nanoparticle probes. Insome embodiments, the nanoparticle probes can be used for celltargeting. In some embodiments, cells are targeted based on thethickness of their glycocalyx.

In some embodiments, the nanoparticle probe comprises one or more of ananoparticle base structure and a slip bond moiety. In some embodiments,the nanoparticle probe comprises one or more of a nanoparticle basestructure and a high affinity moiety. In some embodiments, the slip bondmoiety is configured to form reversible bonds to a target structure of acell. In some embodiments, the slip bond moiety's target structure is oris on the surface of a glycocalyx of the cell (e.g., a surface protein;glycoprotein, saccharide, etc.). In some embodiments, the high affinitymoiety preferentially binds to a target structure of the cell at thecell surface. In some embodiments, the nanoparticle probe binds to thetarget cell at least in part based on its accessibility to the cellsurface or the thickness of a glycocalyx layer of the cell.

Any of the embodiments described above, or described elsewhere herein,can include one or more of the following features.

In some embodiments, the nanoparticle probe further comprises a tetherfunctionalized to the nanoparticle base structure and to the slip bondand/or the high affinity moiety (e.g., an associative moiety). In someembodiments, the high affinity moiety binds strongly and/or irreversiblyto a cell surface protein, etc. In some embodiments, the tether forms aconnection from the nanoparticle base structure to the slip bond and/orhigh affinity moiety.

In some embodiments, the slip bond has a binding strength of less thanabout 100 pN.

In some embodiments, the high affinity moiety has a binding strength oflarger than 100 pN.

Some embodiments pertain to methods of diagnosing disease states. Someembodiments pertain to methods of diagnosing dysfunctional tissue. Insome embodiments, the method of diagnosing dysfunctional tissue in apatient comprises administering the nanoparticle probes described aboveto the patient. In some embodiments, the method of diagnosingdysfunctional tissue comprises detecting a nanoparticle probe in apatient.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the nanoparticle probes disclosed herein are describedbelow with reference to the drawings of certain embodiments. Theillustrated embodiments are intended to demonstrate, but not to limit,the present disclosure. The proportions and relative dimensions andsizes of each component as shown in these drawings forms part of thesupporting disclosure of this specification, but should not be limitingon the scope of this specification, except to the extent that suchproportions, dimensions, or sizes are included in any individual claims.The drawings contain the following Figures:

FIG. 1A-B show a schematic of a circulating tumor cell.

FIG. 2 shows a fluid shear stress model in endothelial cells liningblood vessels.

FIG. 3 shows an embodiment of targeting nanoparticle.

FIG. 4 shows the attachment of the embodiment of FIG. 3 to a cell.

FIGS. 5A-C show another embodiment of a targeting nanoparticle.

FIG. 6A-C show electron microscope images of EC glycocalyx and shedding.

FIG. 7A shows a design for DNA origami (prosthetic) with sequence andcrossovers.

FIGS. 7B-7F show embodiments of possible DNA origami constructs.

FIGS. 8A and 8B show flow diagrams of bioreactors.

FIG. 9 shows an embodiment of a nanoparticle probe.

FIG. 10 shows various embodiments, self-assembling structures.

FIGS. 11A, 11B, and 11C show theoretical images obtained usingembodiments of the nanoparticle probes disclosed herein.

FIGS. 12A and 12B shows an embodiment of a targeting nanoparticle withtether length that changes over time. FIG. 12A shows the startingtargeting nanoparticle. FIG. 12B shows the degradation of a coating overthe core of the nanoparticle after time has passed.

DETAILED DESCRIPTION

Some embodiments of the present disclosure pertain to nanoparticleprobes, methods of making the same, and methods of using the same forbiological targeting and analyses. Some embodiments disclosed hereinpertain to nanoparticle probes (and methods for manufacturing and usingthe same) having features that allow them to differentiate betweencells. In some embodiments, the nanoparticle probes described herein candifferentiate groups of cells by variations in the glycocalyx layers ofthose cells. While several embodiments are discussed below in referenceto “nanoparticles” or “nanoparticle probes,” it should also beunderstood that, while the prefix “nano” is used (e.g., having adimension and/or diameter of equal or less than about 1 nm to about 1000nm), this prefix is illustrative. It should be understood that thefeatures and improvements described herein are also applicable tomicroprobes and microparticles (e.g., having a dimension and/or diametergreater than or equal to about 1 μm to about 100 μm). Any features,structure, or step disclosed herein can be omitted. While theillustrated examples include features, these features need not bepresent in all embodiments. Further, for purposes of summarizing thedisclosure, certain aspects, advantages, and features of the inventionshave been described herein. However, not all embodiments include orachieve any or all of those aspects, advantages, and features.

Non-communicable diseases, such as Type 2 diabetes, hypertension, andchronic kidney disease, are a leading cause of death and disabilityworldwide. Without a definite cure and due to the multi-causal nature ofthe diseases, modern medicine is limited in treating these diseasestates. For example, unless early prevention is possible, medicine iscapable of only treatment of symptoms. In rare cases though, remissionwithout relapse can occur in response to exercise, diet, and mechanicalstimulation-based therapies. This result suggests that the biologicalmechanisms of disease treatment can offer successful health outcomes forpatients. If the mechanisms behind self-repair can be probed andunderstood, these biological responses can be bolstered, facilitated,and/or stimulated, treating of the underlying cause of the disease. Forthat reason, elucidation of the biological mechanisms of disease maylead to novel therapies. In some embodiments, the nanoparticle probesdisclosed herein allow early diagnosis of the above disease statesand/or diagnosis before acute symptoms occur.

It has been noted that there is currently a lack of technology thateffectively investigates the biological phenomena and biologicalprocesses (e.g., outcomes) underlying these disease states. Someembodiments described herein provide diagnostic methods and devices forprobing biological systems. In some embodiments, the techniques anddevices disclosed herein can probe biological systems to betterunderstand the disease, disease progression, and/or disease treatment.In some embodiments, the techniques and devices can be used toeffectively treat the disease. In some embodiments, nanoparticle probesare used to accomplish these outcomes (e.g., to investigate biologicalsystems, etc.). In some embodiments, by probing biological systems usingnanoparticle probes, treatment and/or elucidation of diseases (e.g., byunderstanding the mechanisms underlying a disease state or disorder) canbe accomplished. In some embodiments, biological mechanisms of diseasestates and disorders can be elucidated and the development of noveltreatment methods for disease can be developed. Some embodiments pertainto facilitating biological responses and/or treating of the underlyingcause of the disease using nanoparticle probes.

Some embodiments described herein pertain to nanoparticle probes thatdifferentiate between groups of cells. In some embodiments, healthycells can be targeted using the nanoparticle probes disclosed herein. Insome embodiments, non-healthy cells can be targeted using thenanoparticle probes disclosed herein. In some embodiments, celltargeting is achieved by interactions between the nanoparticle probesdisclosed herein and moieties at the cell surface (or in the cell). Insome embodiments, the nanoparticle probes bind reversibly and/orirreversibly to target cells. In some embodiments, the cellsurface-binding is based on associations between the nanoparticle probesand cell moieties (e.g., motifs, cell surface ligands, cell surfacemarkers, cell surface receptors, surface proteins etc.).

In some embodiments, nanoparticle probes target cells based on one ormore properties of the cell's glycocalyx. The glycocalyx is a layer thatcovers the surfaces of various cell types. It is comprised ofglycoproteins and other carbohydrate-based moieties. The glycocalyxcomposition and structure varies depending on the cell and, in somecases, whether the cell is in a state of disease or not. For instance,the thickness of certain glycocalyx layers can range from less than 100nm to several micrometers, depending on the cell type and/or thesurrounding conditions (e.g. salt excess result in coalescing of certainglycans, leading to EC glycocalyx shrinkage and stiffening, which ispotentially associated with endothelial dysfunction). In someembodiments, variations in cell glycocalyx layers can be exploited fornanoparticle probe targeting by using tailored binding motifs of thenanoparticle probe.

In some embodiments, the nanoparticles disclosed herein can befunctionalized (e.g., through covalent bonds, binding, etc.) withcell-targeting ligands. In some embodiments, the nanoparticle isfunctionalized with one or more of folate, sialyl Lewis X, biotin,streptavidin, RGD, DNA and/or RNA origami, and/or combinations thereof.In some embodiments, the nanoparticle probes comprise ligands that bindto one or more cell surface targets including inflammatory markers (e.g.E-selectin), cell surface glycoproteins (e.g. ICAM-1), and/ormechanoreceptors (e.g. CD29).

The thickness of a cell's glycocalyx is an important biomarker ofchronic inflammation. The glycocalyx layer covers the surfaces ofvarious cell types and is comprised of glycoproteins and othercarbohydrate-based moieties. The glycocalyx thickness can range fromless than 100 nm to several micrometers (e.g., about 1 μm to about 10 μmor about 100 μm). The thickness of the glycocalyx depends on the celltype and surrounding environmental conditions. The overall structureacts as a mechanosensor that physically detects the surrounding fluidshear stress (FSS) through FSS-dependent integrins, syndecans, primarycilia, and other mechanoreceptors. Mechanical deformation results indownstream signaling and activation of shear responsive elements. Insome embodiments, these mechanosensory properties can be exploited andtargeted using the nanoparticle probes functionalized with selectablebinding groups, as described elsewhere herein.

The glycocalyx of endothelial cells (EC) behaves in a somewhat uniquefashion. For example, the EC glycocalyx is capable of adaptiveremodeling over time. Additionally, in certain environments, theglycocalyx of EC cells thins compared to other cell types or compared toother EC cells in different environments. In inflammatory and/or hypoxicconditions, such as is found in diabetic patients, various factors(e.g., C-reactive protein, reactive oxygen species, advanced glycationend-products, etc.) can lead to the degradation or thinning of the ECglycocalyx. In some circumstances, this thinning is found incapillaries, though it may be noted in various blood vessel structures.ECs in low FSS regions, such as capillaries, express a thin glycocalyx,whereas ECs in high FSS regions, such as arteries, express a thickerglycocalyx. Mechanosensitivity can be dependent on multiple factors,including glycocalyx composition, thickness, and other variables, suchas cross-linkages. Ignoring the biological and mechanical complexity ofthe glycocalyx and mechanoreceptors, some of which is still underinvestigation or unknown, the following general observations are made:(1) a thinner glycocalyx increases mechanosensor sensitivity; (2) whenthe glycocalyx thickness decreases to a point where the glycocalyx is nolonger intact (e.g. deglycanated fibers do not induce sufficienthydrodynamic drag force to pull on FSS mechanoreceptors, such as apicalB 1 integrins), there will be reduced mechanosensitivity, (3) in veryhigh FSS, found in certain pathophysiological conditions, glycocalyxshedding dominates, resulting in either a thinner glycocalyx or ECdenudation from shear injury.

In some circumstances, the glycocalyx thickness is greater than thelength of the tether and the nanoparticle probe does not bind toadhesion receptors on a cell surface. FIG. 1A demonstrates physiologicalconditions, where glycocalyx thickness exceeds the length of adhesionreceptors and most surface receptors (not shown). In some circumstances,the glycocalyx thickness is less than the length of the tether and thenanoparticle probe does bind to adhesion receptors on a cell surface.FIG. 1B displays a pathophysiological state, where low FSS,inflammation, and hypoxic conditions result in glycocalyx shedding, thusincreasing the chance for tumor cell binding during metastasis viaexposure of integrin receptors (˜11 nm height). The spacing betweencarbohydrate complexes or glycans are typically 20 nm apart. In someembodiments, the nanoparticle probes bind to these cells and can be usedto identify and/or deliver therapeutics to these cells.

Additionally, there is an association between low basal chronicinflammation, low FSS, and glycocalyx degradation (i.e., thinning of theglycocalyx/thickness reduction) in ECs. Flow and mass transfer areimportant in biological systems. Systemic fluid flow not onlyencompasses the cardiovascular, lymphatic, and primo-vascular system,but also includes interstitial fluid and fascia for signaling andmaintenance. When fluid flows along a material, there is a perpendicularforce or FSS along the contacted boundary surface area, and shear rateis the velocity gradient dependent on the fluid viscosity and FSS. FSSin the microcirculation occurs on EC surfaces along the walls ofcapillaries and other vessels. Plasma viscosity affects mass transfer,and there is a strong correlation between increased viscosity or“stickiness” in biological fluids and various pathophysiologicalconditions. FIG. 2 shows a fluid shear stress model in endothelial cellslining blood vessels. Fluid shear stress is the force experiencedperpendicular to the contacted EC surface area (π=F/A). Shear rate isexpressed by the change in velocity between two flow layers (γ=dz/dy).The diameter of capillaries can be as small as 3 μm. Capillary densityis about 600/mm³, equivalent to about 40 μm between adjacent capillariesof about 1 mm length.

Cell properties, such as changes in glycocalyx thickness, can bepathophysiological indicators of disease (e.g., an early indicator priorto acute symptoms). In some embodiments, the nanoparticle probesdisclosed herein target cells in environments demonstrating low basalchronic inflammation, low fluid shear stress (FSS), and cells withglycocalyx degradation. In some embodiments, the nanoparticle probesdescribed herein associate with cells having thinning glycocalyx layers(e.g., by associating with one or more adhesion receptors of the cell)or with thinning glycocalyx layers themselves. In some embodiments, thepresence of a disease state or a type of disease state itself can bedetermined by measuring the thickness of the glycocalyx. For example, insome embodiments the nanoparticle probes (e.g., functionalizednanoparticles, etc.) are able to target cells (e.g., diseased cells orcells indicating the presence of a disease state, etc.) based on theglycocalyx thickness. In some embodiments, the nanoparticle probesselectively bind to cells based on whether they have a glycocalyx above,equal to, or below a certain thickness. In some embodiments, thenanoparticles target cells having a glycocalyx below a targetedthickness threshold. In some embodiments, because the surrounding flowproperties of fluid around a cell can determine the thickness of acell's glycocalyx, the nanoparticle probes are able to indicate and/ortarget cells depending on the flow properties their environment. In someembodiments, the nanoparticle probes can target cells with glycocalyxthickness (e.g., measured from the cell surface to the terminus oraverage terminus of the glycocalyx) of less than or equal to about: 5nm, 15 nm, 50 nm, 100 nm, or ranges including and/or spanning theaforementioned values. In some embodiments, the early pathophysiology ofparenchyma can be detected with this method.

In some embodiments, the nanoparticle probes adhere to cells that residein specific flow conditions (e.g., fluid shear stress conditions) and donot adhere to cells in other flow conditions. For instance, in someembodiments, the nanoparticle probes adhere to cells residing in anenvironment at a certain minimum fluid shear stress or flow rate. Insome embodiments, the nanoparticle probes adhere to cells residing in anenvironment where the fluid shear stress is less than or equal to about:0.01, 0.1, 1, 5, 10 dynes/cm², or ranges including and/or spanning theaforementioned values. In some embodiments, the nanoparticle probesadhere to cells residing in an environment where flow rate less than orequal to about is about 0.01, 0.02, 0.05, 0.10, 0.20, 0.50 mm/sec, orranges including and/or spanning the aforementioned values. In someembodiments, detachment of the nanoparticle probe to the cell target inflow conditions is dependent on changes in on-off binding rate due toincreasing hydrodynamic drag or increased holding/unbinding force. Insome embodiments, detachment of the nanoparticle probe occurs at theblood-endothelial interface where fluid velocity is increased nearcapillary walls. In some embodiments, the nanoparticle probes adhere tocells residing in an environment at a certain maximum fluid shearstress. In some embodiments, nanoparticle probes adhere to cellsresiding in an environment where the maximum fluid shear stress is lessthan or equal to about 10, 15, 20 dynes/cm², or ranges including and/orspanning the aforementioned values. In some embodiments, the maximumflow rate is about 0.50, 1, 3, 5, 7 mm/sec, or ranges including and/orspanning the aforementioned values.

Nanoparticle probes can be trapped in collapsed capillaries (ifcapillaries were initially opened from increased blood circulation butis followed by pressure lower than the interstitial fluid). Minimum FSSranges in capillaries may be present in local hypoxic regions, due toinsufficient blood flow from increased fibrinogen content. In someembodiments, nanoparticle probes capable of accessing interstitial fluidflow can buildup in low FSS regions of the interstitial fluid. MinimumFSS ranges in larger vessels are found in aneurysms. Maximum FSS rangesin larger vessels result in endothelial denudation from shear injury.

While targeting and detecting cells with variable glycocalyx thicknessesto investigate and/or treat disease states is one potential use of thenanoparticle probes described herein, these nanoparticle probes can beused in a variety of applications. For instance, the technologiesdescribed herein find application in theranostic nanomedicine,regenerative medicine, tissue engineering, and basic research inmechanotransduction. In some embodiments, the proposed technology canhave a large impact in both medicine (e.g., medical treatment of diseaseand diagnosis) and diagnostic research (e.g., research in in vitro andin vivo applications).

The nanoparticle probes can have a variety of configurations that allowit to bind to target cells. In some embodiments, the nanoparticle probecomprises one or more of a nanoparticle base structure (e.g., ananoparticle, a core, etc.), a tether (e.g., an extension that canprotrude from the nanoparticle), a hinge, and/or an associative moiety.In some embodiments, the nanoparticle probe lacks one or more of ananoparticle base structure (e.g., a nanoparticle, a core, etc.), atether (e.g., an extension that can protrude from the nanoparticle), ahinge, and/or an associative moiety.

As shown in FIG. 3, the nanoparticle probe can comprise one or more of acellular prosthetic, a hinge region, a base structure with a weakaffinity ligand (e.g., an antibody), a tether, and an associative moiety(e.g., CD29, scFv, sdAb, etc.). In some embodiments, the nanoparticleprobe does not comprise one or more of the cellular prosthetic, hingeregion, base structure with a weak affinity ligand (e.g., an antibody),tether, and/or the associative moiety (e.g., CD29, scFv, sdAb, etc.). Insome embodiments, as shown in FIGS. 3 and 4, the base comprises a weakaffinity ligand (e.g., a slip bonding moiety, etc.). In someembodiments, the slip bond allows the nanoparticle probe to attach anddetach from cells. In this way, the slip bond allows the strongerbinding associative moiety to bind to the cell surface where, forinstance, the tether is of sufficient length to penetrate the glycocalyxcompletely. Thus, the nanoparticle probe can reversibly bind via theslip bond until it reaches a particular cell where the glycocalyx has anappropriate depth to allow the associative ligand to bind. As shown inFIG. 4, where the nanoparticle nanoprobe binds to a portion of theglycocalyx in a manner that the associative moiety (e.g., the CD29,scFv, sdAb, irreversible binding agent, etc.) does not contact itstarget cell surface marker, the slip bond will eventually release thenanoparticle probe to bind to another cell. In some embodiments, theslip bond allows flow through the plasma (or another biological medium)until the nanoparticle probe associative moiety binds to a cell surface.In some embodiments, the associative moiety binds strongly to aparticular, pre-selected cell surface ligand based on the target cellmarker that is expressed, thereby targeting a particular diseased cell(for diagnosis or delivery of a therapeutic). In some embodiments, asdiscussed elsewhere herein, the slip bond forms a transient bond thatallows the associative moiety to bind to the cell surface depending onthe length of the tether. In some embodiments, the slip moiety can actas a targeting and/or binding portion of the nanoparticle probe. In someembodiments, the associative moiety forms irreversible bonds to a celltarget feature.

FIG. 5A-C show an alternative embodiment of a nanoparticle probe. Asshown, in some embodiments, the nanoparticle probe can comprise aplurality of strong binding associative moieties (e.g., 1, 2, 3, 4, ormore) coupled to the base via a plurality of tethers (e.g., 1, 2, 3, 4,or more, respectively). In some embodiments, as shown, the nanoparticleprobe can comprise a plurality of slip bonds (e.g., 1, 2, 3, 4, ormore). In some embodiments (not pictured) as discussed elsewhere herein,a base structure is not required and the associative moieties and slipbonds are simply located on a tether.

As shown in FIG. 5, the hinge can link the base portion to ananoparticle group that facilitates travel and binding of thenanoparticle nanoprobe to a cell surface. In some embodiments, the hingeallows rotational freedom. In some embodiments, the nanoparticle groupcreates drag and/or directional orientation of the nanoparticlenanoprobe to facilitate detachment from the cell surface. In someembodiments, the cellular prosthetic (and/or nanoparticle group) createsadditional drag to modulate mechanoreceptor sensitivity. In someembodiments, the hinge gives the nanoparticle probe rotational freedomand/or flexibility. In some embodiments, the hinge of FIG. 4 forexample, can give the nanoparticle probe rotational freedom and/orflexibility but is conformationally restricted to prevent totally freemovement about the hinge. In some embodiments, the hinge allows thecellular prosthetic to travel in the direction of fluid flow whileallowing the slip bond and/or the associative bond to properly align tobind to the cell surface.

In some embodiments, the nanoparticle probe of FIG. 3 can be used forglycocalyx-dependent targeting for delivery of mechanosensors orcellular prosthetic to microvascular ECs for tissue engineeringapplications, in order to lower the FSS threshold of apicalmechanoreceptors ‘in vitro’ (not in scale). FIG. 4 shows the attachmentof the embodiment of FIG. 3 to a cell having a thin glycocalyx. On thefigure, the tether is unlabeled, and located between a scFv/sdAb andbase.

In some embodiments, the embodiment of FIG. 5A-C can be used, forexample, for early in vivo imaging and delivery to microvascular ECsnearby inflamed regions (not in scale; position is relative).

In some embodiments, the nanoparticle probes described herein utilizeone or more of hydrodynamic drag, slip bond behavior, tether length,convection/diffusion behavior, and changes in cell membrane fluidity inresponse to fluid shear stress (FSS) as a way to target cells. In someembodiments, this allows cells with a glycocalyx below a specifiedthickness or cells in specific FSS conditions to be targeted. In someembodiments, static or pulsed magnetic field can alter cell membranefluidity and cell phenotype.

Due to the effect of size on hydrodynamic drag, nanoparticles arediffusion dominant, compared to microparticles that are convectiondominant. In other words, the shape and size of nanoparticle probes hasan effect on their biodistribution, determined by their diffusivity(i.e., Stokes-Einstein-Sutherland equation), convection-diffusionbehavior (i.e., Peclet' s number), and hydrodynamic drag. For instance,assuming a Peclet number (convection:diffusion)=1, in the smallestcapillary (about 3 μm), in order for convection to partially dominate inthe lower limits of fluid flow in a perfusion bioreactor (about 1-10μm/sec), the nanoparticle probe can have a diameter ranging from about20 to about 160 nm. In some embodiments, the nanoparticle probe diameter(or a largest dimension of the nanoparticle probe) is greater than equalto about: 20 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm,or ranges including and/or spanning the aforementioned values. In someembodiments, the nanoparticle probe diameter (or a largest dimension ofthe nanoparticle probe) is less than equal to about: 20 nm, about 50 nm,about 100 nm, about 150 nm, about 200 nm, or ranges including and/orspanning the aforementioned values. Since some embodiments of thetargeting technology incorporate glycocalyx surface slip bonds andtethering across the glycocalyx, the hydrodynamic drag is characterizedby two different states, which can be characterized by computationalmodeling of nanoparticle probes in free flow, prior to binding, and oftethered objects near surfaces, after binding to a surface. In someembodiments, the targeting feature simulates a convection-dependent andglycocalyx-thickness-dependent biodistribution within a biologicalsystem.

In some embodiments, as mentioned above, the nanoparticle does notcomprise one or more of a base/scaffold structure, a tether, a hinge, oran associative moiety. For instance, in some embodiments, a series oftethers, as discussed elsewhere herein, are bound together at a nucleuswithout a nanoparticle core. In some embodiments, single functionalizedtether can operate as the nanoparticle probe. In some embodiments, thetether is not present and associative moieties are bound directly to ahinge or a nanoparticle to provide a nanoparticle probe. Any combinationis envisioned of nanoparticles, tethers, hinges, and associativemoieties is envisioned.

In some embodiments, by virtue of any one of shape, physical properties,electrostatic properties, and/or targeting ligands, a nanoparticle probecan target a cell. In some embodiments, the nanoparticle shape can beselected based on its hydrodynamic drag in flow ranges of interest. Insome embodiments, the nanoparticle's on-off binding within capillarypulsatile flow ranges, with attachment below a FSS threshold ofinterest, is used for flow-dependent microvascular targeting andassessment of microvascular perfusion to study disease models or shiftin environment, states, or other stimuli.

In some embodiments, the nanoparticle and/or nanoparticle probes can begenerally and/or substantially spherical in shape, rod-shaped,disc-shaped, cube-shaped, or otherwise. In some embodiments, thenanoparticle probe can have a flat or low curvature base structure thatcan be used. In some embodiments, the length is of a certain ratio tothe height or width of the nanoparticle probe. In some embodiments, thelength and width of the nanoparticle probe have a ratio wherein theratio is greater than about 1.5:1, about 2:1, about 3:1, about 4:1,about 5:1, ratios ranging between two of the aforementioned ratios, orotherwise. In some embodiments, the length and height of thenanoparticle probe have a ratio wherein the ratio is greater than about1.5:1, about 2:1, about 3:1, about 4:1, about 5:1, ratios rangingbetween two of the aforementioned ratios, or otherwise.

In some embodiments, the materials of the nanoparticle (e.g., thebase-structure, the cellular prosthetic, the hinge, nanoparticle groupetc.) can be selected to prevent penetration through glycocalyx (e.g.,hydrophobic, anionic, etc.). In some embodiments, the nanoparticle (ornanoparticle probe) comprises one or more materials be selected toassociate to the cell surface. In some embodiments, the nanoparticlecomprises one or more of DNA and/or RNA origami or another nanomaterial(e.g., a paramagnetic nanoparticle, quantum dot, nanocrystal). In someembodiments, the nanoparticle base structure comprises one or more ofgold or iron-oxide core nanoparticle or other nanoparticles, not limitedto single-chain polymer nanoparticles (e.g., PEG, PGA, PGA-g-PEG, PLA,PLGA, etc., having molecular weights of at least about: 10,000 g/mol,100,000 g/mol, 200,000 g/mol, or ranges spanning the aforementionedvalues) and poly(amidoamine) (PAMAM) dendrimers. In some embodiments,the nanoparticle base structure comprises one or more of colloidal gold,TNF-bound colloidal gold, albumin (or other similar biomolecules),dendrimeric poly(l-lysine), dendrimeric poly(l-lysine) which presentsanionic, naphthalene disulphonate surface groups, dendrimericpolypropylenimine (PPI), Denkewalter-type PLL dendrimer, Tomalia-typePAMAM dendrimer, hydroxylated PAMAM dendrimer, Hult-type poly(ester)(bis-MPA) dendrimer, Majoral/Caminadetype phosphorous-based dendrimer,Simanek-type triazine based dendrimer, Jayaraman/Jain-typepoly(propyletherimine) (PETIM) dendrimer, PEG-PLL, PEG-PAMAM, PETIM-DG,PEG-PPI, peptide dendrimer conjugate, and polystyrene latex particles.

In some embodiments, the nanoparticle probe comprises a neutral ornegative zeta potential. In some embodiments, the nanoparticle probecomprises a fluorophore or other functional component.

In some embodiments, the particle size (e.g., a diameter of theparticle, a largest dimension of the particle, etc.) of the nanoparticleprobe (or microparticle probe as the case may be) is less than about 10μm, about 5 μm, about 1 μm, about 900 nm, about 800 nm, about 700 nm,about 600 nm, about 500 nm, about 400 nm, about 300 nm, about 200 nm,about 100 nm, about 50 nm, about 25 nm, about 10 nm, ranges spanning theaforementioned values, or otherwise.

In some embodiments, as discussed elsewhere herein, the base structurecan be functionalized with slip bonds to allow increased bindingaffinity of the associative moiety. In some embodiments, the slip bondsallow increased binding affinity of the associative moiety due toincreased residency time for the binding of the associative moiety. Insome embodiments, reversibly associative moieties—those capable offorming slip bonds (e.g., low-affinity antibodies with K_(d)>10⁻⁷ M orligands)—are functionalized (e.g., covalently, through binding,coupling, complexation, hydrophobic/hydrophilic interactions, ionically,etc.) to the base (e.g., a nanoparticle) to target cells. In someembodiments, binding affinity can be increased by multivalency (as shownin FIG. 5, for example). In some embodiments, a tether binds the slipbond to the nanoparticle probe, while in other embodiments, a tether isnot used. In some embodiments, slip bonds (e.g., low affinity antibodiesor ligands that have a bind strength below about 100 pN) function as atargeting moiety for temporary binding of nanoparticle probe to aglycocalyx surface target. In some embodiments, slip bonds or non-slipbonds bind to secondary mobile targets on the surface of the glycocalyx.In some embodiments, these slip bonds associate to the surface in low(0.05-1 dyne/cm²) or moderate (10 dyne/cm²) FSS conditions. In someembodiments, the slip bonds dissociate in high (>10 dyne/cm²) FSSconditions. In some embodiments, slip bonds or another targetingmodality for binding to secondary target(s) on glycocalyx surface whereincreased binding efficiency is desired are used. In some embodiments,the non-slip bonds or higher affinity bonds (e.g., the associativemoieties) are used as a permanent or higher duration targeting moiety.In some embodiments, non-slip bonds or higher affinity bonds are used inglycocalyx-thickness-limiting tethers or other component. In someembodiments, the nanoparticles or nanoparticle probes are targeted usingselectins such as PECAM, VCAMs, and ICAMs and integrins such asCD11/CD18 functionalized to the surface of the nanoparticle. In someembodiments, a nanoparticle (DNA and/or RNA origami for example) can bepatterned with either small molecules, antibodies, or larger proteins.In some embodiments, they are patterned with RGD peptides, anti-ICAM VHHantibodies, or sialyl Lewis X.

In some embodiments, the material selected for incorporating orgenerating a slip bond can comprise one or more of an binding motif, aDNA and/or RNA, a dendrimer, or another polymers/nanomaterials with aneutral or negative zeta potential for the base (i.e., base plate). Insome embodiments, the material can be of any shape. In some embodiments,the material is spherical, rod-shaped, disc-shaped, cube-shaped, orotherwise. In some embodiments, the material is a position-limited shape(e.g., flat or low curvature on attachment site) to control theorientation or position of the attached nanoparticle. In someembodiments, the slip bonds include a hyaluronan targeting motif,chondroitin sulfate targeting motif, dermatan sulfate targeting motif,heparan sulfate targeting motif or other glycocalyx surface EC targetingmotifs. In some embodiments, the slip bond are lectin-binding proteinssuch as recombinant CD44, bounded to the material covalently or byspecific binding groups (biotinylation or oligonucleotide conjugation atthe C or N terminus).

In some embodiments, the slip bond moiety can be formed using one ormore of a carbohydrate-based ligand (sialyl Lewis X) or low-affinityantibody isolated via chromatography . In some embodiments, the bindingstrength of the slip bond moiety is below about 500 pN, about 100 pN,about 50 pN, about 10 pN, about 5 pN, ranges spanning those values orotherwise. In some embodiments, the binding strength of the slip bondmoiety is at least about 0.5 pN, about 1 pN, about 5 pN, ranges spanningthose values or otherwise. In some embodiments, the slip bond is formedin conjunction with a cytoskeletal-independent (i.e., mobile) target onthe cell's glycocalyx surface. In some embodiments, the dissociationconstant of the slip bond moiety is at least about 10⁻³ M, which isaround the dissociation constant of sialyl Lewis X to E-selectin (878μM). In some embodiments, the binding strength of the associative moietyis at least about 2000 pN, 1000 pN, 500 pN, about 100 pN, rangesspanning those values or otherwise. In some embodiments, thedissociation constant of the associative moiety is at least about thoseof strongly binding antibodies, whose dissociation constants range from10⁻¹³ to 10⁻⁷ M. Without being bound to any particular mechanism, it isbelieved that the hydrodynamic drag force on the nanoparticle can causeits dissociation at higher flow rates, and fluid shear stress increasescell membrane fluidity (affects mobile component). In some embodiments,this feature modifies the nanoparticle to become moreconvection-dependent. In some embodiments, increased binding time at lowflow rates increases the binding efficiency of the tether, if within thetarget thickness threshold.

In some embodiments, slip bonds that dissociate under increased FSS(e.g., above about more than 1 dyne/cm², with a binding strength below50 pN (which is the hydrodynamic drag at which Brownian motion isnegligible for a microparticle) are used to fabricate the nanoparticleprobe. In some embodiments, the slip bond (e.g., weak affinity antibodyor ligand) dissociates at higher FSS and increases binding time at lowerFSS (pulsatile).

In some embodiments, the slip bond comprises a biodegradable ordetachable section for eventual isolation of tethered prostheticconstruct. In some embodiments, this isolation allows removinguntethered nanoparticles that are bounded to its target solely by slipbonds, due to low FSS environment relative to target. In someembodiments, linkage of additional tether(s) to other functionalproducts (e.g., enzymes, drugs, ligands, imaging agents (e.g., dyes),bases for self-assembly nanoparticles, tissue engineering initiators)can be performed.

In some embodiments, for example, where a base structure is not used,slip bonds may be placed between spacing of tethers. In someembodiments, slip bonds may be placed between spacing of tethers where abase structure is used.

As stated above, tethers can be used in addition to or instead ofnanoparticle probe base structures. In some embodiments, as discussedelsewhere herein, a tethered nanoparticle group induces hydrodynamicdrag in flow conditions (see FIG. 5). In some embodiments, the tetheracts as a targeting moiety for binding to a primary target on a cellsurface. In some embodiments, the tether acts as a targeting moiety forbinding to a primary target on a cell surface through a glycocalyx belowa specified thickness, whose attachment is limited by tether length. Insome embodiments, tethers function as spacers between the slip bondmoiety and the nanoparticle core.

In some embodiments, a tether can have variable dimensions. In someembodiments, the diameter of the tether, for instance, varies by target.In some embodiments, if targeting ECs, diameter can be selected to beless than about 18 nm to penetrate the glycocalyx. In some embodiments,the diameter can be selected to be less than about 50 nm, about 25 nm,about 20 nm, about 15 nm, about 10 nm, about 5 nm, ranges spanning theaforementioned values, or otherwise to, for instance, penetrateglycocalyx. In some embodiments, the diameter is about 18 nm, topenetrate glycocalyx based on the distance between carbohydrate brushstructures of the glycocalyx. In some embodiments, the length of thetether can vary. In some embodiments, the length varies by target. Insome embodiments, if targeting microvascular ECs of capillaries, thetotal tether length can be between about 20-200 nm. In some embodiments,the tether length is below about 400 nm, about 300 nm, about 200 nm,about 100 nm, about 50 nm, about 25 nm, about 10 nm, ranges spanning theaforementioned values, or otherwise. In some embodiments, the length issufficient to reach and bind to a cell surface target through thetargeted cell's glycocalyx.

In some embodiments, where the tether is functionalized to a basestructure, the spacing of the tether on the nanoparticle can be varied.In some embodiments, spacing between carbohydrate complexes or glycansin ECs are assumed to be ubiquitously 20 nm apart. Glycocalyx thicknessin diseased EC capillaries ranges from 10 nm to 30 nm in comparison tothe control range of 60 nm to 80 nm; however, this thickness may be anunderestimation since characterization of the glycocalyx is limited bytechnology and technique. In some embodiments, the tethers are selectedto be of sufficient length to allow binding to the cell surfaces ofdiseased EC capillaries and/or carbohydrate complexes or glycans in ECs.

Tether length and design can be manipulated to increase bindingefficiency to the target. Compared to flexible tethers, controllingdiffusive motion on a rigid hinged tether would have a higher bindingprobability to a nearby target. In some embodiments, the spacing oftethers on, for example, the nanoparticle probe base varies by target.In some embodiments, the spacing is at least about 1 tether/10 nm²,about 1 tether/50 nm², about 1 tether/100 nm², about 1 tether/200 nm²,about 1 tether/300 nm², about 1 tether/400 nm², about 1 tether/600 nm²,or about 1 tether/800 nm². In some embodiments, if targeting ECs, thespacing is at least about 1 tether/400 nm². In some embodiments,multiple tethers can be used together on a single nanoparticle probe. Insome embodiments, for 2 μm microparticles and 500 nm nanoparticles, hassignificantly smaller spacing and shorter tether length. In someembodiments, the overall adhesion of the nanoparticle can be 4-17 foldlower than the microparticles, which may be significantly enhanced bythis invention via slip bonds. In some embodiments, if multiple tethersare used, spacing (<1 tether/400 nm²) above glycocalyx surface can beselected to penetrate glycocalyx.

In some embodiments, the tethers are functionalized at the distal end oftether (scFv or other) to bind against primary target of interest nearcell membrane (receptors of interest, such as selectins and integrins,are ˜11 nm above membrane).

In some embodiments, the material selected for use as a tether cancomprise one or more of DNA and/or RNA, a glycan, a lipid, a glycolipid,a proteoglycan, or other polymers/nanomaterials. In some embodiments,the material selected is a recombinant viral tail fiber or a syntheticpolymer that mimics its properties. In some embodiments, the tether cancomprise polyethylene glycol (PEG) having a molecular weight below about500 Da, 1000 Da, 3000 Da, 10000 Da, etc.. In some embodiments, thematerial is neutral in charge. In some embodiments, the tether has acore structure(s) that is rigid. In some embodiments, the tether is withor without flexible joint(s) to limit diffusive movement of tether inorder to maximize binding chance to cell surface target through theglycocalyx. In some embodiments, despite the negative charge of DNAorigami, its penetration of the glycocalyx is successful. In someembodiments, this is due to a 15 nm×100 nm, 24 double helix bundle DNAorigami rod that can penetrate HUVEC glycocalyx in static conditions.

In some embodiments, the tether comprises a targeting moiety. In someembodiments, the targeting moiety comprises one or more of an antibody,antibody fragment, ligand to cell surface receptor (ie. Sialyl Lex),aptamer, receptor, or other targeting entities.

In some embodiments, the hydrodynamic drag of tethered nanoparticlestakes into account other forces such as lift or buoyancy, and tetherednanoparticles (less than about 80 nm in size) have a contradictoryhydrodynamic drag of 1-2 pN at 2 mm/sec flow velocity based on a studyof nanoparticles tethered to E-toxin receptors having a measured springconstant of 0.7 pN/μM after flow. Normal blood flow velocity is greaterthan about 1 mm/sec in the microvascular beds, and would be much lowernear the wall, with exclusion of areas near the compression for redblood cells (RBCs) which would have an increased shear rate. Mean RBCflow velocity can range from 0.2 to 0.8 mm/sec in capillaries.Furthermore, pulsatile flow exists in capillary beds. Thus, if a slipbond strength with, for instance, a binding force below about 10 pN isused, biodegradable or detachable section for isolation of tetheredprosthetic construct, for the purpose of removing untetherednanoparticles that are bounded to its target solely by slip bonds, dueto low FSS environment relative to target may become redundant (but thismay depend on non-EC targets).

In some embodiments, the general design of glycocalyx-dependenttargeting technology rely on a tethered antibody (monoclonalsingle-chain variable fragment) against a primary target. In someembodiments, a tether(s) of a specified length is bounded to a basestructure containing slip bonds to a secondary target if higher affinityis desired, and complexed with a hinge and nanobody. In someembodiments, slip bonds dissociate under increased FSS, with a bindingstrength below 50 pN. In some embodiments, the nanobody of a designedshape and deformable material, will induce hydrodynamic drag (>50 pN)after temporary binding to components of the glycocalyx surface via slipbonds (˜1-10 pN binding strength) under flow conditions. In someembodiments, this property provides the nanoparticle probe constructwith unique convection-dependent behavior. In some embodiments, thedimensions are determined for convection to primarily dominate within aselected flow velocity range, as previously mentioned. In someembodiments, when temporarily bounded through slip bonds, thehydrodynamic drag experienced by the construct in flow conditions may beresisted by tether forces due to cell membrane fluidity. Cell membranefluidity is associated with membrane viscosity, and FSS increases ECmembrane fluidity. In some embodiments, the time between slip andrelease affects tethered antibody binding chance to cells below aglycocalyx-thickness threshold. In some embodiments, this slip-bondfeature simulates flow-dependent targeting in the nano-scale. However,in some embodiments, non-specific binding will occur near static regionsif hydrodynamic drag is insufficient to break the slip bonds. In someembodiments, the number and spacing between tethers can be determined.

FIGS. 12A and 12B show an embodiment of a nanoparticle probe with tetherlength that varies over time. In some embodiments, as shown in FIG. 12A,the nanoparticle probe has a core that is coated with a biodegradablelayer. In some embodiments, as the outer biodegradable layer thins overtime, the tethers become more exposed (as shown in FIG. 12B), and lengthof the tethers relative from the surface is increased. In someembodiments, the tethers are encapsulated by a biodegradable material toachieve this change. In some embodiments, the biodegradable material iscomprised of one or more of poly-D-L-lactide-co-glycolide, polylacticacid, poly-ε-caprolactone, chitosan, gelatin, orpoly-alkylcyanoacrylates. In some embodiments, this degradation andexposure allows the nanoparticle probe to initially bind to cells with aseverely degraded glycocalyx (e.g., via a short tether). Later, aftersome degradation, the nanoparticle probe is able to bind to cells with amoderately degraded glycocalyx. In some embodiments, the encapsulatingmaterial surpasses the length of the tether, or partially encapsulatesthe length of the tether. In some embodiments, this negates the need fordesigning multiple nanoparticle probes with a specific tether length foradjustment in detection. In some embodiments, time-lapse imaging can beused to map out region-specific severity or accessibility to a cellsurface by tether length.

FIG. 6 shows electron microscopic views of the hearts stained to revealthe glycocalyx. (A) An intact glycocalyx after 20 min of perfusion(Group E) and (B) the nearly completely degraded endothelial glycocalyxafter 20 min of warm ischaemia and consecutive reperfusion (Group A).(C) The glycocalyx after pre-treatment with antithrombin and 20 min ofwarm ischaemia and reperfusion (Group B). The endothelial glycocalyx ismostly intact. In some embodiments, the nanoparticle probes disclosedherein can be varied to target any one of the glycocalyx structures ofFIGS. 6A-C to the exclusion of or together with the others.

In some embodiments, as discussed elsewhere herein, the nanoparticle canbe attached to a hinge component. In some embodiments, the attachment ofnanoparticle to a hinge above glycocalyx allows (or facilitates)computational modeling of hydrodynamic drag of tethered nanoparticles.In some embodiments, this modeling is for future modifications of theconstruct. Nanoparticle groups may be elevated above the base portion ofthe nanoparticle probe via hinge to facilitate computational modelingfor future modifications. For in vivo applications, in some embodiments,the length of the nanoparticle probe tether and hinge accounts forcompression by RBCs on the entire component, which may be addressed bydistance to the base during compression. Shape and surface properties ofthe nanoparticle probe may be modified to alter hydrodynamic drag basedon the application.

In some embodiments, the nanoparticle groups (as shown in FIG. 5A-C) areused as a scaffold for targeting cells. In some embodiments, forexample, a nanoparticle or nano-construct is selected based on itshydrodynamic drag properties. In some embodiments, these reversiblebinders can be functionalized to a nanoparticle by one or more tethers.In some embodiments, functionalized tethers of a specific length ordimension are selected. In some embodiments, the selection of a tetheris based on its ability to restrict binding to cells with a glycocalyxbelow a certain thickness (or that have some other targetable quality).In some embodiments, the selection of a tether is based on its abilityto bind to a primary target.

In some embodiments, the hydrodynamic drag of a nanoparticle that isattached to a base structure, which increases binding affinity for thenanoparticle. The design of the embodiment varies based on theapplication.

In some embodiments, drag forces of interest include those that are atleast about 500 pN, about 100 pN, about 50 pN, about 10 pN, about 5 pN,ranges spanning those values or otherwise. In some embodiments, the dragforce of interest include is at least about 50 pN for most physiologicalflow rates. In some embodiments, the hydrodynamic drag of thenanoparticles disclosed herein varies by distance to a surface of thecell, due to introduction of other forces such as lift.

In some embodiments, computational modeling for tethered cells can beperformed, but this modeling is not applicable to nanoparticles.However, in some embodiments, a 30-50 nm diameter nanoparticle have 1-2pN hydrodynamic drag at 0.2 cm/s. Using alternative materials, such asDNA origami (patented by Harvard) for its hydrophilicity andelectrostatic repulsion against the glycocalyx, has not been attemptedto my knowledge.

In some embodiments, design parameters are based on the probe'shydrodynamic drag, slip-bond strength, and convection-diffusionbehavior. In some embodiments, prior to conducting complex studieswithin a living body, the nanoparticle probe is designed in a way toallow it to be modeled outside the body in a simple yet alternateapplication—resolving a limitation in tissue engineering. To addressinsufficient cell growth in areas of slow media flow that occurs evenwith sufficient nutrients, mature vessels will be stabilized in low flowconditions, and vessel sprouting will be stimulated in near staticregions. This can be achieved within a large scaffold through deliveryof mechanosensor prosthetics to endothelial cells (cells lining theblood vessels) located in low flow conditions and characterized by athin glycocalyx. In some embodiments, cell surface modification willlower the mechanical threshold at which cells sense fluid flow in theirsurrounding environment, which creates a shift in the physiologicaldevelopment of blood vessels. In some embodiments, a more robustpre-vascularized construct can be developed, thereby addressing alimiting factor in the engineering of various tissues.

Some embodiments pertain to developing a targeting technology onmicrovascular endothelial cells. In some embodiments, the technology canbe used in a simple 2-D cell culture flow model, under several flowconditions and simulated pathophysiological states. In some embodiments,testing can then be conducted in a complex 3-D flow model as a pilotstudy for further investigation.

In some embodiments, the nanoparticle probes described herein haveapplication in the fields of theranostic nanomedicine, regenerativemedicine, tissue engineering, and basic research in mechanotransduction.In some embodiments, the nanoparticle probes allow users to explorebiological mechanisms that occur during the remission ofnon-communicable chronic diseases, the leading cause of death anddisability in the United States. In some embodiments, this approach canlead to the advent of novel diagnostic methods and therapies. In someembodiments, the nanoparticle probes can be used in a method ofdiagnosing non-communicable chronic disease and/or the stage of progressof that disease. In some embodiments, the nanoparticle probes candiagnose “at risk” individuals prior to that patient showing symptoms ofthe disease. In some embodiments, the nanoparticle probes can be used todevelop treatment strategies and therapeutic regimens for patients. Forexample, in some embodiments, the nanoparticle probes can be used todetermine if a particular treatment regimen is suitable (e.g., diet andexercise versus drug treatment, etc.). There is an association betweenmicrocirculatory dysfunction in the development and pathogenesis ofvarious chronic diseases, including diabetes, hypertension, end-stagerenal disease, osteoporosis, and other chronic conditions. However,there is no robust strategy to target or monitor subtle early changes indeep tissue regions with poor microcirculatory status for measuringintervention outcome in translational research. In some embodiments, thenanoparticle probes described herein can be used in theranosticnanomedicine.

FSS induces glycan synthesis in ECs, and the EC glycocalyx is degradedin inflammatory conditions, especially in diabetes. FSS induces glycansynthesis in ECs, yet FSS also causes glycocalyx shedding, therebycounteracting each other to establish a balance. The EC glycocalyxfunctions as a mechanosensor that detects the surrounding FSS and iscapable of adaptive remodeling over time. Thus, in some embodiments,controlled targeting of ECs characterized by a degraded glycocalyx inlow FSS by the nanoparticle probes disclosed herein has a variety ofpotential applications.

In some embodiments, the nanoparticle probes can be used to lower theFSS detection threshold of mechanoreceptors. In some embodiments,increasing the hydrodynamic drag force experienced by mechanoreceptors,such as integrins (e.g., (β1 Integrin subunit), syndecans, primarycilia, and other mechanosensors, has various biomedical applications(e.g., in tissue engineering). In some embodiments, the (β1 Integrinsubunit (CD29) in ECs is characterized by deglycanated mechanoreceptorcomplexes. FSS-induced CD29 is involved in various signal transductions,including elongation, angiogenic signaling, and EC reorientation ofcardiomyocytes. Vascular endothelial growth factor (VEGF) and FSS playsa synergistic role in capillary morphogenesis, and FSS attenuatessprouting while VEGF gradients direct sprouting. Lowering the FSSdetection threshold of CD29 in apical ECs would stabilize mature vesselsin lower FSS regions and stimulate sprouting in near-static regions,potentially yielding a more robust prevascularized in vitro 3D constructfor tissue engineering applications. DNA origami is a potentialprosthetic material, due to electrostatic repulsion to the glycocalyx,its biodegradability by nucleases prior to tissue transplantation toavoid immunogenicity, and non-toxicity if intracellularized with itstarget and degraded by lysosomes. In some embodiments, the nanoparticleprobes can be used for tissue engineering strategies. In someembodiments, the nanoparticle probes can be used for vasculardevelopment for to various tissues, such as the bone, heart and otherorgans and or for tissue development of bone, heart and otherorgans/tissues. The development of a robust prevascularized constructremains a limiting factor on sufficient stem cell growth within the coreof large scaffolds and other matrices in top-down approaches of tissueengineering. In some embodiments, the nanoparticle nanoprobes allowtargeted vascular development and growth. In some embodiments, one ormore agents bound to the nanoparticle nanoprobe include VEGF,angiopoietins, or FGF.

The vascular endothelium is an early site of damage during inflammation.Thus, in some embodiments, using the nanoparticle probes could allow thepreservation of the endothelial surface layer of microcirculatoryvessels under microvascular dysfunction and could reduce damage causedby either chronic low-grade inflammation or reperfusion. In someembodiments, the nanoparticle probes could detect and or target an areaof disease leading to the diagnosis and treatment of the disease usingthe nanoparticle probe as a delivery agent for, for instance,pharmaceuticals or other small molecules. As a diagnostic agent, thenanoparticle probe can be used to indicate early patient pathology andcan be used to develop a treatment regimen (e.g., medicines/supplementsto be used, dietary changes, exercise regimens, etc.). In someembodiments, adverse effects on physiological processes can be limitedor avoided if the nanoparticle design (tether length) limits its bindingto cells below a specified glycocalyx thickness of interest.

In some embodiments, FSS-induced CD29 in ECs is involved in varioussignal transductions, including elongation, angiogenic signaling, and ECreorientation of cardiomyocytes. Vascular endothelial growth factor(VEGF) and FSS play a synergistic role in capillary morphogenesis, andFSS attenuates sprouting while VEGF gradients direct sprouting. In someembodiments, lowering the FSS detection threshold of CD29 in apical ECswould stabilize mature vessels in lower FSS regions and stimulatesprouting in near-static regions, potentially yielding a more robustprevascularized in vitro 3D construct for tissue engineeringapplications.

In some embodiments, other mechanotransduction-based studies andadditional applications may be obtained through other modificationsincluding attachment of enzymes or other prosthetics to altermechanoreceptor properties or other cell functions, and these cellulartargets would not be limited to ECs. There are over 300 cell types inhumans. In some embodiments, markers of any one of these cells can betargets of the ligands of the nanoparticle probes disclosed herein. Insome embodiments, in vitro applications can be established to refine thenanoparticle probe targeting technology for conducting in vivo researchof various animal disease models.

In some embodiments, the nanoparticle probe utilizes one or more ofhydrodynamic drag, slip bond behavior, tether length,convection/diffusion behavior, and changes in cell membrane fluidity inresponse to fluid shear stress (FSS), in order to target cells below aspecified glycocalyx thickness and in low FSS conditions. Theassociation between low basal chronic inflammation to low FSS andglycocalyx degradation in ECs is an early pathophysiological indicator.In vitro applications can be initially established as a blueprint, priorto conducting in vivo research. In some embodiments, the nanoparticleprobes can be used to validate the design parameters of model systemsfor an in vitro study. In some embodiments, the nanoparticle probe andsystems using the same can accomplish at least the following specificaims: 1) Developing a Design in a 2-D Cell Culture Flow Model and 2)Validating Design in a Perfusion Bioreactor.

These aims can be accomplished by validating the nanoparticle probe'stargeting properties (FSS/glycocalyx thickness-dependent targeting) andits functional modification (targeted delivery of mechanosensorprosthetics on the cell surface) for potential tissue engineeringapplications. In some embodiments, the approach involves conductingexperiments in a microbioreactor under different flow rates of cellmedia and pathophysiological conditions that affect glycocalyxthickness. In some embodiments, the nanoparticle probe can be used tolower the mechanical threshold at which microvascular endothelial cellssense fluid flow in their surrounding environment. In some embodiments,this is achieved through attachment of a prosthetic molecules to anepitope of interest on a mechanoreceptor. In some embodiments, thenanoparticle probes could be used to experimentally validate the inducedphenotypic changes and the targeting properties of the nanoparticleprobe. In some embodiments, this is achieved through measurement ofintracellular metabolites or indicator dyes. In some embodiments,testing systems and use of nanoparticle probes with glycocalyx-thicknessand FSS-dependent targeting properties can be developed. In someembodiments, testing systems are organs-on-chips.

In some embodiments, the nanoparticle probes can be used to addressinsufficient cell growth in areas of low flow that occurs even withsufficient nutrients by improving vascularization techniques in largescaffolds. In some embodiments, this is achieved through delivery ofmechanosensor prosthetics. Experiments in perfusion bioreactorco-culture under different flow rates can be performed to controlbiodistribution. The nanoparticle probes could be used to experimentallyvalidate stabilized mature vessels in low flow conditions and vesselsprouting in near static regions. Changes can be associated with a 3-Dflow profile, with velocimetry techniques. In some embodiments, testingsystems and use of nanoparticle probes could identify areas where thistechnology could be used to address limitations in tissue engineering.In some embodiments, cell types are tested for potentialglycocalyx-thickness-dependent targetability or for differences inglycocalyx surface targets or characterization.

In some embodiments, the nanoparticle probes could be used in tissueengineering design. For instance, in some embodiments, such as bonetissue engineering, perfusion bioreactors could be used. Perfusionbioreactors have improved the mass transfer of nutrients and growth ofFSS-dependent stem cells seeded in bone scaffolds. Perfusion bioreactorsare typically characterized as a closed system containing one or moretissue constructs, and having an inlet and outlet for media flow.

For in vitro applications, control of mechanical stimulation is lackingin larger 3D cell culture constructs. Despite improvements, cellulardensity near the core is less than the periphery. Flow variability fromfrictional loss within bioreactors may lead to insufficient growth inareas of low FSS, even with sufficient nutrients. In other cases,insufficient nutrients will result in a necrotic core. While variousadvancements have been made in bone tissue engineering, such as magneticcomposite scaffolds and vascularization techniques, that address thesevarious problems, there remains much room for improvement to fullyaddress current challenges in tissue engineering. Tissue engineeringstrategies for vascular development can be applied to other tissues,including the heart, and other organs (kidney, liver, lung, intestines,pancreas, stomach, etc.). The development of a robust prevascularizedconstruct remains a limiting factor on sufficient stem cell growthwithin the core of scaffolds and other matrices in tissue engineering.

In some embodiments, strategies in diagnostic imaging to determinemicrocirculatory status can be performed using nanoparticle probes. Insome embodiments, as discussed elsewhere herein, the nanoparticle probescan be used in in vivo applications. The development of chronicpathophysiology may be a result of an imbalance between the amount ofdamage and the rate at which the damage is addressed, e.g. removal ofinsult by leukocytes and repair by stem cells. In addition tomesenchymal stem cells (MSCs), these stem cells include endothelialprogenitor cells (EPCs) deriving from the bone marrow that are overallresponsible for vascular repair and maintains the integrity of themicrocirculation especially through physical exercise. FSS and cytokinesinteract to control the concentration of E-selectin surface expressionin ECs. Damage to endothelium can be associated with an increase incirculating ECs from EC denudation or glycocalyx shedding due to veryhigh FSS or other insults. Endothelial damage is also associated with adegraded EC glycocalyx in low FSS regions from inflammation that resultsin increased intravascular adhesion by leukocytes and platelets.However, the glycocalyx maintained in postcapillary EC venules are eventhicker in low FSS and inflammation. The ability to visualize or targetcapillary regions with early indications of poor microcirculatory statususing the disclosed nanoparticle probes, could be used as a theranostictool in a variety of chronic disease animal models. In some embodiments,nanoparticle probes with an affinity for low FSS conditions and thinglycocalyx would improve upon the sensitivity of potential vascularpreclinical detection methods, such as glyconanoparticles inneuroinflammation. In some embodiments, the pericyte/endothelialinterface is of interest.

Glycocalyx thickness in diseased EC capillaries ranges from 10 nm to 30nm in comparison to the control range of 60 nm to 80 nm. Technology todeliver functional nanoparticle probes to cells in low FSS within acertain glycocalyx-thickness threshold have various applications,including, but not limited to: diagnostic imaging (e.g., ofmicrocirculatory status), delivery of nanoparticles for therapeuticeffects in areas of microcirculatory dysfunction, and delivery ofmechanosensor prosthetics to lower FSS detection by mechanoreceptors.

In some embodiments, imaging modalities or other non-invasivealternatives could be developed or incorporated. Current vascularimaging technologies include PET, SPECT, MRI, intravascular ultrasound,and optical imaging via optical coherance tomography or near-infrared(NIR) fluorescence. Fluorescence molecular tomography (FMT) is a form of3-dimensional imaging of NIR fluorescent probes capable of deep-tissuevisualization and early disease or biomarker detection via targetedapproach, compatible with smart probes. In some embodiments, thenanoparticle probe can be functionalized with one or more dyes or radioopaque agents for visualization. In some embodiments, the nanoparticleprobe can be functionalized with one or more of dyes including but notlimited to indocyanine green (ICG), methylene blue, Cy5.5, ZW800-1, andIRDy 800CW. In some embodiments, the nanoparticle probe described hereincould be functionalized with FMT agents. Complementing other modalities,FMT of degraded EC glycocalyx of capillaries in low FSS would serve as ageneralized yet sensitive inflammatory indicator of early pathogenesisin nearby tissues, which carries a diagnostic advantage over currentapproaches in development that have a smaller disease spectrum due todisease-specific targets. In some embodiments, NIR quantum dots (QDs)and other dyes or proteins, capable of deep-tissue visualization andearly disease or biomarker detection can be implemented in the targetedapproach described herein (as well as using nanoparticle probes with oras smart probes). Complementing other modalities, FMT of degraded ECglycocalyx of capillaries in low FSS would serve as a generalized yetsensitive inflammatory indicator of early pathogenesis in nearbytissues, which carries a diagnostic advantage over current approaches indevelopment that have a smaller disease spectrum due to disease-specifictargets. Other approaches include magnetic nanoparticle probes for MRIimaging, and post-isolation of tissue for regional characterization inanimal studies. In some embodiments, characterization can be associatedwith invasive diagnostics, such as blood collection devices andminimally invasive nanotainers.

FIGS. 11A, 11B, and 11C show imaging of a brain that could be achievedusing the disclosed nanoparticle probes. Glyconanoparticle distributionin (A) Control, (B) Multiple Sclerosis, and (C) Stroke animals modelsused for disease detection or diagnosis of neuroinflammation.Glyconanoparticles are functionalized with sialyl-LeX, which is acarbohydrate ligand that has an affinity to E-selectin, which isupregulated during inflammation.

Delivery of Nanoparticles for Therapeutic Effects in Areas ofMicrocirculatory Dysfunction: FSS/glycocalyx-dependent targeting appliedto carriers can enhance the therapeutic delivery of agents, such assiRNA and biologics to microvascular ECs in low FSS conditionscharacterized by a thin glycocalyx layer. In some embodiments, thistargeted region is a pathophysiological site for treatment strategies.Furthermore, in some embodiments, nanoparticle probes have a dimensionof at least 15 nm to not undergo capillary diffusion unless in cancer.

Delivery of Mechanosensor Prosthetics to Lower FSS Detection ofMechanoreceptors: The vascular endothelium is an early site of damageduring inflammation. Thus, lowering FSS threshold detection wouldpreserve the endothelial surface layer of microcirculatory vessels undermicrovascular dysfunction and would reduce damage caused by eitherchronic low-grade inflammation or reperfusion. Adverse effects onphysiological processes can be limited or avoided if the nanoparticleprobe design (tether dimensions, possibly <100 nm length and <15 nmdiameter) limits its binding to cells below a specified glycocalyxthickness of interest.

In some embodiments, the nanoparticle probes described herein, havingFSS/glycocalyx-thickness-dependent cell surface targeting, would notonly expand vascularization strategies in tissue engineering, but alsocould be used to develop an alternative mechanism to enhance theeffectiveness of cell-based therapeutics.

In theranostic applications, a generalized yet sensitive region-specificinflammatory/viscous composite biomarker such as those described hereinwould lead to further advances in the field of screening and earlydetection of various chronic conditions, when treatments are moreviable. Furthermore, in some instances, identifying regions with earlyinflammatory damage in the microcirculation is more advantageous forinvestigating integrative medicine, where regions with subtleimprovements can be identified for high-throughput analysis and wherealternative non-invasive diagnostic approaches can be improved, such asproteomics or metabolomics of skin surface collections. In someembodiments, the nanoparticle probes are used to facilitate theinvestigation of pathways involved in the high remission rate (with rarecases of relapse) of various chronic diseases despite poor prognosis,induced by a non-traumatic region-specific pulsatile blunt forcetargeting observed in a private practice.

From potentially addressing the source rather than the symptom ofvarious chronic conditions, the mechanotransduction-based therapy, insome embodiments, is likely mediated through deformation of theextracellular matrix (ECM) that leads to downstream synergisticbiological responses, such as stem cell activation and/or viscousalterations in hemodynamics or interstitial fluid dynamics. In someembodiments, the following uses of nanoparticle probes are possible: (1)simulating this biological phenomenon by altering cellular response tothe surrounding FSS and by targeting ECs characterized by a degradedglycocalyx in pathophysiology, (2) creating a novel translational toolto explore this new field, and (3) accounting for the potential role(s)of the primo vascular system involved in stem cell transport andinvestigating epigenetic mechanisms.

To reduce FSS threshold of CD29 in ECs with a thin glycocalyx, ananti-CD29 nanobody (sdAb) or single chain variable fragment (scFv)functionalized DNA-origami prosthetic is designed. Taking advantage ofthe negative charge of DNA, sterical hindrance, and tether length of thenanobody (<100 nm), the designed prosthetic has a higher affinity totargeted receptors of ECs characterized by a glycocalyx below aspecified thickness. The parameters of the prosthetic will be determinedbased on pilot studies on its functions, such as eliciting anappropriate drag force in low FSS conditions. The construct willincorporate a functionalized dendrimer base or other structural basethat utilizes a low-affinity slip bond, against EC glycocalyx surfacedeterminants. This feature would increase the carrier's affinity to theglycocalyx if necessary and would not interfere with the nanoparticleprobe's mechanical mechanism for CD29 stimulation, since the secondarytarget would not restrict CD29 activation in response to FSS-inducedhydrodynamic drag. With the base, in high FSS conditions, the increasein cell membrane fluidity lowers binding time, in contrast to low FSSconditions. The primary objective of this application is to improvecurrent vascularization techniques in tissue engineering. Additionaldesigns and applications depend on the desired goal of the project.

Therapeutic strategies and other methodologies targeting the glycocalyxwith non-specific alterations (which include stem cell coatings andproteoglycan mimetics) are unable to achieve the specific purpose ofthis proposed application. This is due to the presence of an intactglycocalyx, which prevents accessibility to the site of interest.Furthermore, a degraded glycocalyx, with deglycanated fibronectinbounded to CD29, or a very thick glycocalyx would reduce the sensitivityof the mechanosensor, which necessitates a more targeted approach.Furthermore, there is no simple pharmaceutical drug to achieveFSS-induced responses due to the physical activating mechanism behindmechanoreceptors. Manipulation via magnetic nanoparticle probes andcomposite scaffolds can achieve these responses; however, these methodshave a potential issue with biocompatibility and toxicity that wouldneed to be addressed in the future, and are FSS-independent methods.

In some embodiments, project feasibility is dependent on knowndimensions and properties of cells in the microcirculation to determinethe parameters most useful to develop the proposed technology. In theglycocalyx of ECs, the spacing between glycans (GAGs) can be about 20nm, and is assumed to be ubiquitous. Glycocalyx thickness in diseased ECcapillaries range from 10 nm to 30 nm in comparison to the control rangeof 60 nm to 80 nm, but these measurements are region-dependent. CD29 onthe EC apical surface mediates FSS signaling, and the extracellulardomain of CD29 has a height of 11 nm, which is about the span ofselectins and other integrin receptors. Based on mAb-magnetic beadstudies, CD29 activation occurs with 1.2 pN pulling force incardiomyocytes and 200 pN in MSCs. In some embodiments, a thresholdbelow 1.2 pN in ECs can be used since EC glycocalyx thickness is closeto cardiomyocytes and should have similar mechanoreceptor properties. Insome embodiments, a force of 130 pN, which is equivalent to ˜10% of dragforce experienced by a 30 μm×30 μm EC with FSS of 12 dynes/cm², resultsin integrin mechanosensory complex activation. Thus, in someembodiments, the EC CD29 threshold would need to be verified forrefining modeling parameters. Laminar flow is essential, since twistingCD29 in ECs, which simulates turbulent flow, will result in a differentsignal transduction. In some embodiments, the average superficial flowvelocity of bone grafts in bioreactors is about 0.06 cm/s, ranging from0.0001 to 0.15 cm/s in different regions. In some embodiments, thenanoparticle probes have a dimension of at least 15 nm to not undergocapillary diffusion unless in cancer.

In some embodiments, the dimension of an Fab fragment of an IgG antibody(nanobody) is about 8 nm×4 nm×5 nm. In some embodiments, the rationalefor the general size of the nanoparticle probe can be is as follows: fora Peclet number (convection:diffusion)=1, in a small capillary (˜3 μm),in order for convection to partially dominate in the lower limits offluid flow in the bioreactor (˜1-10 μm/sec), the nanoparticle probe canhave a diameter ranging from ˜20 - 160 nm.

In some embodiments, a model to estimate the drag force on the DNAorigami can be used. Previous attempts to model hydrodynamic drag ontethered DNA strands using statistical mechanics have been verifiedexperimentally by holding one end of the DNA with optical tweezers andexposing the other end to hydrodynamic flow. However, DNA origamibundles have increased stiffness therefore making bending contributionsnegligible. Moreover, in some embodiments, it would be more accurate tomodel the entire DNA origami but not accurate to consider the effect ofhydrodynamic drag on a known material with approximately the samegeometry, such as a solid cone.

In some embodiments, hydrodynamic drag force is often modeled usingStoke's drag force expression: F_(d)=6πC_(shape)C_(tether)ηrv, where ηis the coefficient of viscosity, r the radius of the particle (radius ofcone), and v the velocity of the fluid (assuming the tethered particlehas no velocity). C_(shape) and C_(tether) are correction coefficientsfor the shape of the DNA origami and the fixed end, as Stoke's dragforce expression originally assumes spherical particles in free flow. Insome embodiments, an empirical expression of C_(shape) (also known asthe dynamic shape factor) for axisymmetric shapes, such as a cone, withmotion normal to the axis of symmetry: C_(shape)=0.392+0.621S−0.040S2,where S is the ratio of the surface area of the cone, divided by thesurface area of a sphere that has the same perimeter as the sideperimeter of the cone. In some embodiments, a tethering correctioncoefficient developed can be used:

$= {\sum\limits_{i = 0}^{N}{c_{i}h^{- 1}}}$

where h is the ratio of the cone radius and length, and c_(i) are fittedcoefficients.

However, in some embodiments, this is much larger than the actual forceon tethered DNA origami.

In some embodiments, antibody detachment threshold is unlikely to occurin using the following parameters. In some embodiments, 250 pN on ananti-CD29 mAb results in 0% antibody detachment, and 350 pN results in15% detachment. In some embodiments, the epitope of interest or selectedmAb is selected so that it does not interfere with CD29 function, sinceattachment at functional sites such as hinge regions could prevent CD29activation. Other mAb types include activating CD29 mAbs that targetactivated CD29 and prevent its deactivation, and for clarification,cannot bind to non-active CD29. Thus, in some embodiments, an anti-CD29mAb can be selected to avoid these potential issues.

Engineering of sdAbs are commercially available by Creative-Biolabs, andoriented conjugation of sdAbs to nanoparticle probes are feasible as analternate application via C-terminus attachment of 6-His-Cys bymutagenesis. Specific attachment of oligonucleotides to the C-terminusof sdAbs or other proteins can be achieved. Functionalization of DNAorigami structures with this product and protocols for increased yieldafter purification can be done using synthetic techniques. In someembodiments, DNA origami design/modeling of 3D nanostructures and theirsynthesis can follow standardized protocols, and commercial services arealso available through Tilibit Nanosystems. Base rupture is unlikely(dG/dC ˜20pN; dA/dT ˜14pN; stacking 2pN). Lyosomal degradation of DNAnanodevices following apical CD29 internalization or recycling in ECs,if it occurs, would pose no problem for the immune system followingtransplantation, unless antigen presenting ECs are capable of presentingthe degraded product as an antigen detectable by DNA receptors; however,this can be addressed with nucleases. Inactivation, removal, or absenceof nuclease in media typically present in serum would ensure in vitrostability of the DNA nanodevice, and this non-permanent feature can beused to ensure degradation of DNA-exposed products prior totransplantation. Much progress have been made in translating thistechnology to the cell. Recent progress have been made for DNA origamistability in serum.

Although dendrimer synthesis is well-established, in some embodiments alow affinity ligand, sialyl Lex, has 50pN binding strength to selectinsites with a fast off-on rate that varies by force and ramp rate. Insome embodiments, finding a weak or weaker low-affinity ligand or analogto glycocalyx targets is a potential concern. However, in someembodiments, an alternative solution is using weak-affinity antibodies,whose isolation will be feasible with commercial purification kits usedfor conducting affinity chromatography. In some embodiments, the degreein which membrane fluidity affects the time for CD29 to bind viadragging of the slip bond is unknown and its effect on nanoparticleprobe function can be validated. In some embodiments, synergisticsignaling among mechanosensors in response to FSS will not be accountedfor in order to reduce the complexity of the research design.Determining if low flow regions in bioreactors have sufficient nutrientscan be investigated, in addition to monitoring the surrounding celldensity. In some embodiments, stabilizing mature vessels in lower FSSregions and stimulating sprouting in near-static regions in a 3Dperfusion bioreactor may make use of additional approaches, such asalterations in media, to utilize the phenotypic change to develop arobust prevascularized construct.

In some embodiments, nanoparticle probe size/shape can be determinedusing: optical tweezers near a surface and nanoparticle trackinganalysis. In some embodiments, the velocity conditions at which brownianforce outweighs the drag force to drive the designed nanoparticle probenear a vessel wall can also be investigated. These parameters can bebased on variations of the rudimentary prosthetic design (FIG. 7), inorder to determine the ideal dimensions prior to conducting futurestudies.

In some embodiments, potential targets include vascularized tissueengineering. Vascularized bone tissue engineering generally utilize aco-culture of MSCs and EPCs, and it is assumed that stem cells express athick glycocalyx and reside in a low FSS niche. Whether differentiatedcell types, such as osteocytes and pericytes, exhibit a thinning of theglycocalyx in low FSS conditions is yet unknown. Although this is apotential concern, it is interesting to note that preliminary studies,suggest that shear stress induces vasoprotective gene upregulation inpericytes. Furthermore, osteocytes regulates bone reformation inresponse to FSS-induced CD29, and would be either beneficial or havelittle effect on the project's application. If MSCs are targeted, thoughunlikely due to their probable glycocalyx thickness, similarCD29-induced FSS responses and ECM interactions and responses wouldapply.

Targeting of cell types other than ECs could occur and, in someembodiments, exploited. In some instances, stem cells express a thickglycocalyx and reside in a low FSS niche. Increased FSS inducesproliferation and differentiation in adult stem cells, but inducesproliferation for most differentiated cells. In some embodiments,differentiated cell types, such as pericytes and osteocytes, exhibit athinning of the glycocalyx in low FSS conditions or have a limitedglycocalyx thickness range and can be targeted. Shear stress couldinduce vasoprotective gene upregulation in pericytes. Furthermore, FSScontrols bone remodeling in osteocytes. Modifications can includeattachment of bioactive molecules (enzymes, ligands, etc) and/orfunctional nanobodies (quantum dots, magnetic nanoparticles, DNA and/orRNA origami, niosomes, liposomes, polymeric nanoparticles, etc) forvarious applications. Thus based on available knowledge on glycocalyxthicknesses, in some embodiments, biomedical applications formicrovascular endothelial cells are feasible. In some embodiments,targeting can be done for any of these cell types but is not limited tothese cell types.

FIG. 7A shows a design for DNA origami (prosthetic) with sequence andcrossovers. Sequence design of scaffold and synthesis of staple strandscan be based off known methods. The proposed design is comprised of a24,210 base scaffold with 730 staple strands, and has the approximatedimensions: cylindrical core length (314bp, ˜100nm), cone length (220bp,˜75 nm), diameter (44-50 nm), inner diameter (˜26 nm). The hollowstructure permits extracellular debris (>6 nm) to coalesce with higherfrequency after tethering to a target, but depends upon theelectrostatic attraction between the DNA and debris. This increases thepotential hydrodynamic drag of the prosthetic in low FSS conditions.

FIGS. 7B-7F show models of DNA origami to target epitopes hidden by theEC glycocalyx. The DNA origami construct is linked to a fluorescentoligonucleotide-QD from Gene Link, connected via single flexible tetherat the linkage site labeled in red. A VHH anti-ICAM1 antibodybiotinylated at the C-terminus is purchased from Creative Biolabs. ICAM1was selected as a target due to commercial availability. Theexperimental design has the antibody conjugated to the tip of each ofthe 3 tethers. For the control design, the antibody is above the body ofthe structure, opposing the tethers. Despite the negative charge of DNAorigami, penetration may not be an issue, since a 15 nm×100 nm, 24double helix bundle DNA origami rod, singly penetrates the glycocalyx ofHUVECs in static conditions in contrast to its single helix controldesign. In some embodiments, the protrusions and/or tethers penetratethe glycocalyx more easily than the body of the structure. In someembodiments, this modification improves the limit of detection forinflammation by vascular-targeting nanoparticles. The nucleotidesequence of the FIG. 7F construct is as follows SEQ ID: 1:

1 aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 61atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 121cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta 181gttgcatatt taaaacatgt tgagctacag caccagattc agcaattaag ctctaagcca 241tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 301ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag 361tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 421cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 481tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 541aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 601ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt 661aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg 721atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 781tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 841caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttc 901tcgtcagggc aagccttatt cactgaatga gcagctttgt tacgttgatt tgggtaatga 961atatccggtt cttgtcaaga ttactcttga tgaaggtcag ccagcctatg cgcctggtct 1021gtacaccgtt catctgtcct ctttcaaagt tggtcagttc ggttccctta tgattgaccg 1081tctgcgcctc gttccggcta agtaacatgg agcaggtcgc ggatttcgac acaatttatc 1141aggcgatgat acaaatctcc gttgtacttt gtttcgcgct tggtataatc gctgggggtc 1201aaagatgagt gttttagtgt attctttcgc ctctttcgtt ttaggttggt gccttcgtag 1261tggcattacg tattttaccc gtttaatgga aacttcctca tgaaaaagtc tttagtcctc 1321aaagcctctg tagccgttgc taccctcgtt ccgatgctgt ctttcgctgc tgagggtgac 1381gatcccgcaa aagcggcctt taactccctg caagcctcag cgaccgaata tatcggttat 1441gcgtgggcga tggttgttgt cattgtcggc gcaactatcg gtatcaagct gtttaagaaa 1501ttcacctcga aagcaagctg ataaaccgat acaattaaag gctccttttg gagccttttt 1561ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtt gttcctttct 1621attctcactc cgctgaaact gttgaaagtt gtttagcaaa accccataca gaaaattcat 1681ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggttgtc 1741tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt tacggtacat 1801gggttcctat tgggcttgct atccctgaaa atgagggtgg tggctctgag ggtggcggtt 1861ctgagggtgg cggttctgag ggtggcggta ctaaacctcc tgagtacggt gatacaccta 1921ttccgggcta tacttatatc aaccctctcg acggcactta tccgcctggt actgagcaaa 1981accccgctaa tcctaatcct tctcttgagg agtctcagcc tcttaatact ttcatgtttc 2041agaataatag gttccgaaat aggcaggggg cattaactgt ttatacgggc actgttactc 2101aaggcactga ccccgttaaa acttattacc agtacactcc tgtatcatca aaagccatgt 2161atgacgctta ctggaacggt aaattcagag actgcgcttt ccattctggc tttaatgaag 2221atccattcgt ttgtgaatat caaggccaat cgtctgacct gcctcaacct cctgtcaatg 2281ctggcggcgg ctctggtggt ggttctggtg gcggctctga gggtggtggc tctgagggtg 2341gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg 2401attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 2461aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 2521ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 2581gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 2641taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 2701ttgtctttag cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat 2761tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt 2821ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg gtattccgtt 2881attattgcgt ttcctcggtt tccttctggt aactttgttc ggctatctgc ttacttttct 2941taaaaagggc ttcggtaaga tagctattgc tatttcattg tttcttgctc ttattattgg 3001gcttaactca attcttgtgg gttatctctc tgatattagc gctcaattac cctctgactt 3061tgttcagggt gttcagttaa ttctcccgtc taatgcgctt ccctgttttt atgttattct 3121ctctgtaaag gctgctattt tcatttttga cgttaaacaa aaaatcgttt cttatttgga 3181ttgggataaa taatatggct gtttattttg taactggcaa attaggctct ggaaagacgc 3241tcgttagcgt tggtaagatt caggataaaa ttgtagctgg gtgcaaaata gcaactaatc 3301ttgatttaag gcttcaaaac ctcccgcaag tcgggaggtt cgctaaaacg cctcgcgttc 3361ttagaatacc ggataagcct tctatatctg atttgcttgc tattgggcgc ggtaatgatt 3421cctacgatga aaataaaaac ggcttgcttg ttctcgatga gtgcggtact tggtttaata 3481cccgttcttg gaatgataag gaaagacagc cgattattga ttggtttcta catgctcgta 3541aattaggatg ggatattatt tttcttgttc aggacttatc tattgttgat aaacaggcgc 3601gttctgcatt agctgaacat gttgtttatt gtcgtcgtct ggacagaatt actttacctt 3661ttgtcggtac tttatattct cttattactg gctcgaaaat gcctctgcct aaattacatg 3721ttggcgttgt taaatatggc gattctcaat taagccctac tgttgagcgt tggctttata 3781ctggtaagaa tttgtataac gcatatgata ctaaacaggc tttttctagt aattatgatt 3841ccggtgttta ttcttattta acgccttatt tatcacacgg tcggtatttc aaaccattaa 3901atttaggtca gaagatgaaa ttaactaaaa tatatttgaa aaagttttct cgcgttcttt 3961gtcttgcgat tggatttgca tcagcattta catatagtta tataacccaa cctaagccgg 4021aggttaaaaa ggtagtctct cagacctatg attttgataa attcactatt gactcttctc 4081agcgtcttaa tctaagctat cgctatgttt tcaaggattc taagggaaaa ttaattaata 4141gcgacgattt acagaagcaa ggttattcac tcacatatat tgatttatgt actgtttcca 4201ttaaaaaagg taattcaaat gaaattgtta aatgtaatta attttgtttt cttgatgttt 4261gtttcatcat cttcttttgc tcaggtaatt gaaatgaata attcgcctct gcgcgatttt 4321gtaacttggt attcaaagca atcaggcgaa tccgttattg tttctcccga tgtaaaaggt 4381actgttactg tatattcatc tgacgttaaa cctgaaaatc tacgcaattt ctttatttct 4441gttttacgtg ctaataattt tgatatggtt ggttcaattc cttccataat tcagaagtat 4501aatccaaaca atcaggatta tattgatgaa ttgccatcat ctgataatca ggaatatgat 4561gataattccg ctccttctgg tggtttcttt gttccgcaaa atgataatgt tactcaaact 4621tttaaaatta ataacgttcg ggcaaaggat ttaatacgag ttgtcgaatt gtttgtaaag 4681tctaatactt ctaaatcctc aaatgtatta tctattgacg gctctaatct attagttgtt 4741agtgcaccta aagatatttt agataacctt cctcaattcc tttctactgt tgatttgcca 4801actgaccaga tattgattga gggtttgata tttgaggttc agcaaggtga tgctttagat 4861ttttcatttg ctgctggctc tcagcgtggc actgttgcag gcggtgttaa tactgaccgc 4921ctcacctctg ttttatcttc tgctggtggt tcgttcggta tttttaatgg cgatgtttta 4981gggctatcag ttcgcgcatt aaagactaat agccattcaa aaatattgtc tgtgccacgt 5041attcttacgc tttcaggtca gaagggttct atctctgttg gccagaatgt cccttttatt 5101actggtcgtg tgactggtga atctgccaat gtaaataatc catttcagac gattgagcgt 5161caaaatgtag gtatttccat gagcgttttt cctgttgcaa tggctggcgg taatattgtt 5221ctggatatta ccagcaaggc cgatagtttg agttcttcta ctcaggcaag tgatgttatt 5281actaatcaaa gaagtattgc tacaacggtt aatttgcgtg atggacagac tcttttactc 5341ggtggcctca ctgattataa aaacacttct caagattctg gcgtaccgtt cctgtctaaa 5401atccctttaa tcggcctcct gtttagctcc cgctctgatt ccaacgagga aagcacgtta 5461tacgtgctcg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 5521tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 5581cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5641ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5701tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5761gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5821tatctcgggc tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa 5881caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc 5941caggcggtga agggcaatca gctgttgccc gtctcgctgg tgaaaagaaa aaccaccctg 6001gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 6061cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 6121cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 6181tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 6241cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg 6301ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 6361atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 6421agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc 6481cggaaagctg gctggagtgc gatcttcctg aggccgatac ggtcgtcgtc ccctcaaact 6541ggcagatgca cggttacgat gcgcccatct acaccaacgt aacctatccc attacggtca 6601atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg 6661atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt cctattggtt 6721aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 6781aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 6841ggtacatatg attgacatgc tagttttacg attaccgttc atcgattctc ttgtttgctc 6901cagactctca ggcaatgacc tgatagcctt tgtagatctc tcaaaaatag ctaccctctc 6961cggcattaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc 7021cggcctttct cacccttttg aatctttacc tacacattac tcaggcattg catttaaaat 7081atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt 7141attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt 7201gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgtt

Delivery of nanoparticle probes can also be used in the delivery ofmatrix-degradation enzymes to reduce flow variability. In someembodiments, degrading the extracellular matrix (ECM) can eliminate lowFSS regions through decreased internal resistance. In some embodiments,this strategy can be used to enhance endothelialization of a tissueconstruct in a time-dependent manner. For example, in some embodiments,carriers functionalized with FSS/glycocalyx-dependent technology can beused to release enzymes at low FSS regions. In some embodiments, thisrelease is followed by enzyme deactivation or removal. Removal can beachieved via magnetic field if enzymes are attached to magnetic NPs. Insome embodiments, ECM remodeling is essential for activation of stemcell niches, but current limitations in technology prevent directremodeling of low FSS regions of a scaffold in a bioreactor.

Delivery of nanoparticle probes can also be used to induce local growthfactor gradient to enhance endothelialization. In some embodiments, thenanoparticle probes can allow specific targeting of low FSS regions. Insome embodiments, this targeting facilitates various strategies increating a local growth factor gradient, such as the incorporation ofthe proposed technology to deliver encapsulated VEGF for endothelialsprouting, thereby enhancing vascularization and addressing similarlimitations in tissue constructs in low FSS regions. The nanoparticleprobes can be functionalized with various active molecules (growthfactors, biomolecules, therapeutics, pharmaceuticals, etc.), which canbe delivered to a site of interest. FIG. 9 shows a functionalizedembodiment of a nanoparticle probe, e.g., a schematic of afunctionalized nanoparticle with encapsulated agents (drugs and nucleicacids). Polymeric biodegradation allows control over release kinetics oftherapeutic agents. Agents can be functionalized to the surface orinside of the nanoparticle probe, diffused within the nanoparticle probe(hydrophobic or hydrophilic associate), or otherwise associated with thenanoparticle probe. In some embodiments, the nanoparticle nanoprobes canbe used to diagnose and treat Type 2 diabetes, hypertension, chronickidney disease, cancer, etc. by functionalizing an effective therapeuticand a targeting moiety to one of the nanoparticle probes describedherein.

In some embodiments, delivery of self-assembly nanoparticles forbottoms-up modification of tissue scaffolds is accomplished. In someembodiments, this approach can include polymer subunits containingcyclic-RGD motifs (or other motifs) for integrin binding to establish anECM. The technology for self-assembly of capillary networks in tissuescaffolds are in its early stages. In some embodiments, the delivery ofself-assembly nanoparticles at low FSS regions may be used in tissueengineering to establish an initial site (a nucleation site) forself-assembly within a scaffold. Several self-assembling particles areshown in FIG. 10 that could be nucleated using the nanoparticle probesdescribed herein. Examples of self-assembly of compounds bearing Phe-Phemotif for the formation of various structures. In some embodiments, thenanoparticle probes can be applied to various self-assembly subunits,such as peptides. In some embodiments, the nanoparticle probes can beapplied to 3D-printed constructs, a bottoms-up tissue engineeringapproach.

In some embodiments, as discussed elsewhere herein, the nanoparticleprobes can be used in in vivo applications. For instance, theassociation among one or more of low basal chronic inflammation, lowFSS, increased viscosity, and glycocalyx degradation in ECs is animportant pathophysiological indicator. These include microcirculatorydysfunction in the pathogenesis of various chronic diseases, includingdiabetes, hypertension, end-stage renal disease, osteoporosis, and otherchronic conditions. The development of chronic pathophysiology is animbalance between the amount of damage and the rate at which the damageis addressed, e.g. removal of insult by leukocytes and repair by adultstem cells and/or pericytes. In addition to mesenchymal stem cells(MSCs), these stem cells include endothelial progenitor cells (EPCs)deriving from the bone marrow that are overall responsible for vascularrepair and maintains the integrity of the microcirculation especiallythrough physical exercise. FSS and cytokines interact to control theconcentration of E-selectin surface expression in ECs. Damage toendothelium is associated with an increase in circulating ECs from ECdenudation or with glycocalyx shedding due to very high FSS or otherinsults. Endothelial damage is also associated with a degraded ECglycocalyx in low FSS regions from inflammation that results inincreased intravascular adhesion by leukocytes and platelets. However,the glycocalyx maintained in postcapillary EC venules are even thickerin low FSS and inflammation. The ability to visualize or targetcapillary regions with early indications of poor microcirculatorystatus, would be useful as a theranostic tool in a variety of chronicdisease animal models. With an affinity for low FSS conditions and thinglycocalyx, it would improve upon the sensitivity of potential vascularpreclinical detection methods, such as glyconanoparticles inneuro-inflammation.

In some embodiments, methods of synthesizing (e.g., preparing) thenanoparticle probes described herein include a step of acquiring ananoparticle base structure. In some embodiments, the nanoparticle basestructure is functionalized with a slip bond. In some embodiments, thenanoparticle is functionalized with a tether. In some embodiments, thetether is functionalized to a slip bond. In some embodiments, the tetheris functionalized to a hinge. In some embodiments, the hinge isfunctionalized to a slip bond. In some embodiments, the nanoparticle isfunctionalized with therapeutic agents. In some embodiments, slip bondsmay be replaced with non-slip bonds.

In some embodiments, the methods described herein involve identifying apatient (e.g., a subject) in need of treatment. In some embodiments, apatient may comprise any type of mammal (e.g., a mammal such as a human,cow, sheep, horse, cat, dog, goat, rodent, etc.). Once identified as apatient, the nanoparticle probe is administered to the patient for aperiod of time. In some embodiments, the period of administrationcomprises a period starting with the diagnosis of a disease state, for aperiod of more than about 1 day, about 2 days, about 3 days, about aweek, about a month, about two months, or until the disease statesubsides. In some embodiments, the nanoparticle probe is administereduntil a time when the disease state is controlled or cured (e.g., theacute symptoms have subsided, symptoms have decreased to a baseline,risk factors death have decreased, etc.), or for a prescribed period oftime of less than about 1 week, about 2 weeks, about 3 weeks, about amonth, about two months, about 6 months, or about a year.

In some embodiments, dosing and delivery of the combination of thenanoparticle probe can be performed for periods between 1 day to fivedays, five days to two weeks, two weeks to a month, a month to twelvemonths. In some embodiments, dosing and delivery of nanoparticle probecan be performed for periods of at least about 1 day, 5 days, 10 days,20 days, 30 days, 50 days, 100 days, 200 days, 300 days, ranges andvalues between the aforementioned values and otherwise.

For purposes of summarizing the disclosure, certain aspects, advantagesand features of the inventions have been described herein. It is to beunderstood that not necessarily any or all such advantages are achievedin accordance with any particular embodiment of the inventions disclosedherein. No aspects of this disclosure are essential or indispensable. Inmany embodiments, the nanoparticle probe system may be configureddifferently than illustrated in the figures or description herein. Forexample, various functionalities provided by the illustrated modules canbe combined, rearranged, added, or deleted. In some embodiments,additional or different processors or modules may perform some or all ofthe functionalities described with reference to the example embodimentdescribed and illustrated in the figures. Many implementation variationsare possible.

EXAMPLES

Based on the inventor's research experience, the following results areprojected using controlled studies.

Example 1

Due to the unavailability of bone marrow microvascular endothelialcells, human dermal microvascular endothelial cells (HDMEC) and mediawill be purchased from Promocell [C-12210, C-39220, C-39215] as analternative. The nanoparticle probe's stability in the cell growth mediawill be validated prior to use, to determine whether countermeasures tostabilize the DNA origami construct or purify the media will benecessary. HDMECs will be flow adapted at 37° C. for 24 hours at 10dynes/cm². A microfluidic system, developed at Johns Hopkins University,that controls oxygen tension and shear stress in cultured cells will beused as described. Cells will be divided into two groups: (1) TreatmentGroup, and (2) Control Group. The control group will utilize eithernanoparticle probe without anti-CD29 functionalization or anti-CD29. Thenanoparticle probe concentration will be based on results from a pilotstudy, whose purpose is to validate the design's overall function. Eachtreatment group will be carried out in five experimental conditionsunder steady flow over 24 hrs: (1) Static conditions [0.01 dyne/cm2, 5%O₂]; (2) Low FSS conditions [0.05-1 dyne/cm², 5% O2]; (3) Physiologicalconditions [10 dyne/cm², 5% O2]; (4) Inflammatory conditions [1dyne/cm², 5% O₂, with infusion of C-reactive protein or an alternativeglycocalyx degradation inducer based on a previous publication; and (5)Ischemic conditions [1 dyne/cm², 1% O₂]. The inflammatory and ischemicconditions at low flow simulates pathophysiological conditions forglycocalyx degradation in the bioreactor environment.

Objective 1: Investigate FSS/Glycocalyx-Thickness-Dependent Targeting inECs

Time-lapse fluorescence will be used to continuously monitornanoparticle probes binding to cells. Time points at 4 hr intervals willbe recorded as average fluorescence intensity per cell and statisticallyanalyzed by repeated ANOVA. Colocalization of labeled nanoparticleprobes and CD29 will be confirmed using fluorescently labeled anti-CD29mAb [EMD Millipore] targeting a different epitope than the nanoparticleprobe CD29 sdAb. Average glycocalyx thickness will be measured by astandardized two-photon laser scanning microscopy method, and comparedamong the groups by ANOVA, to determine whether glycocalyx-thicknessdependent targeting was achieved.

Objective 2: Investigate Lowered FSS Detection Threshold of CD29 in ECs

The biological responses to measure will include endothelial nitricoxide synthase (eNOS) activity and plasminogen activator inhibitor-1(PAI-1) activity. The eNOS activity assay [Cayman Chemicals] and PAIassay [EMD Millipore] will be conducted at the endpoint, following themanufacturer's instructions. Other additional biomarkers, assays, ormethods can be included, but would not be crucial for this proposal.Comparison of activity among groups will be statistically analyzed byANOVA.

Example 2

Conduct Pilot Study on its Applications in Vascularized Bone TissueEngineering

Vascularization studies will be divided into two groups: (1) TreatmentGroup, and (2) Control Group. The bone scaffold will be synthesized as acomposite of nanostructured calcium phosphate cement, and scaffold sizedetermined based on a pilot study on cell density. A co-culture of bonemarrow derived MSCs and peripheral blood derived EPCs with mixed media(bone media with endothelial supplements or endothelial-based media withosteoinductive supplements) will be seeded following protocols, as analternative to the many different vascularization procedures in bonetissue scaffolds. Experiments will be conducted under four steady andpulsatile flow rates (at 0.05 Hz). Flow rate will be determined to yieldan approximate FSS of 0.01, 0.05, 1, and 10 dyne/cm², for static,sub-threshold, and physiological mechanotransductive conditions.Scaffolds will be harvested at 1, 2, and 6 days; and cellular MTTactivity assay will be performed, in addition to histology for measuringcore density by cell type. The 3D velocity field profiles in eachbioreactor condition will be monitored using holographic correlationvelocimetry and then analyzed.

Example 3

Pre-diabetic T2D model using male obese prone rats vs lean rats(age-matched, fed ad libitum and fed-controlled groups) will be comparedusing 3D molecular imaging (IVIS) following intravenous injection ofnanoparticle probes (NIR dyes) to monitor microcirculatory status overtime of interest. Rats will be sacrificed at time points. Tissue will beisolated and homogenized for analysis, and histology of local sites ofinterest will be obtained, in control vs treated rats (treatment may bethrough functionalized nanoparticle probes, or otherpharmaceutical/non-pharmaceutical intervention). Using these testsystems, microcirculatory pathophysiology in the stomach precedingpancreatic damage, in addition to related speculative patterns onconnective tissue (adipose) inflammation, are predicted in the model ofinterest. Providing data on the investigation of potential earlyassociations among various connective tissue - organ systems or local(region-dependent) characterization in various chronic pathophysiologieswill be gathered. At that time early-stage therapeutic strategies forpatients can be developed [including, but not limited to, local deliveryor manipulative methods (e.g. needle-based local guidance signaling ordelivery with ‘smart’ carriers) and integrative practices (e.g.non-traumatic region-specific pulsatile blunt force targeting,transcutaneous electrical nerve stimulation, mind and body manipulation,photo/thermal/magnetic therapies), in addition to addressing safety,repeatability, and reproducibility concerns.

What is claimed is:
 1. A nanoparticle probe comprising: a nanoparticle base structure; and a slip bond moiety configured to reversibly bind to a glycocalyx of a cell; a tether functionalized to the nanoparticle base structure and to an associative moiety configured to bind to a one or more of a cell surface protein, receptor, and/or biomarker of the cell; wherein the associative moiety preferentially binds to the protein, receptor, and/or biomarker of the cell based on the thickness of a glycocalyx layer of the cell.
 2. The nanoparticle probe of claim 1, wherein the slip bond has a binding strength of less than about 100 pN.
 3. The nanoparticle probe of claim 1, wherein the associative moiety has a binding strength of larger than 100 pN.
 4. The nanoparticle probe of claim 1, wherein the associative moiety comprises a ligand for a cell surface receptor and wherein the ligand binds to the cell surface receptor with a binding strength of greater than 500 pN.
 5. The nanoparticle probe of claim 1, wherein the associative moiety comprises one or more of oligonucleotide, RGD, sialyl Lewis X, scFv, and/or sdAb.
 6. The nanoparticle probe of claim 1, wherein the slip bond comprises one or more of a hyaluronan targeting motif, chondroitin sulfate targeting motif, dermatan sulfate targeting motif, heparan sulfate targeting motif, other lectins or glycocalyx surface targeting motifs, a low affinity antibody, and/or combinations of the foregoing.
 7. The nanoparticle probe of claim 1, wherein the nanoparticle base structure comprises a DNA and/or RNA origami, gold nanoparticle, an iron-oxide nanoparticle, poly(amidoamine) (PAMAM) dendrimers, colloidal gold, TNF-bound colloidal gold, albumin, dendrimeric poly(l-lysine), dendrimeric polypropylenimine (PPI), Denkewalter-type PLL dendrimer, Tomalia-type PAMAM dendrimer, hydroxylated PAMAM dendrimer, Hult-type poly(ester) (bis-MPA) dendrimer, Majoral/Caminadetype phosphorous-based dendrimer, Simanek-type triazine based dendrimer, Jayaraman/Jain-type poly(propyletherimine) (PETIM) dendrimer, PEG-PLL, PEG-PAMAM, PETIM-DG, PEG-PPI, peptide dendrimer conjugate, polystyrene latex particles, or combinations thereof.
 8. A method of diagnosing dysfunctional tissue in a patient comprising: administering the nanoparticle probe of claim 1 to the patient; and detecting the nanoparticle probe in the patient.
 9. The method of claim 8, wherein the nanoparticle probe comprises: a nanoparticle base structure; and a slip bond moiety configured to reversibly bind to a glycocalyx of a cell; a tether functionalized to the nanoparticle base structure and to an associative moiety configured to bind to a cell surface protein of the cell; wherein the associative moiety preferentially binds to the cell surface protein of the cell based on the thickness of a glycocalyx layer of the cell.
 10. The method of claim 8, wherein the dysfunctional tissue of the patient is a cancer tissue.
 11. The method of claim 8, wherein the dysfunctional tissue of the patient is caused at least in part by a disease state of the patient.
 12. The method of claim 9, wherein the disease state is type-2 diabetes.
 13. The method of claim 9, wherein the disease state is hypertension.
 14. The method of claim 9, wherein the disease state is chronic kidney disease.
 15. A method of preparing a nanoparticle probe, comprising: coupling a tether to a nanoparticle base structure; coupling a associative moiety to the tether; and coupling a slip bond moiety to the nanoparticle base structure. 